It is common practice for forensic laboratories to quantitate the amount of human genomic DNA recovered from evidentiary biological samples. Motivation for the quantitation of the DNA, which is used as a polymerase chain reaction (PCR) template for multiplex amplification of short tandem repeat (STR) loci, include minimizing the amplification of partial DNA profiles, minimizing allele dropout or imbalance if template amounts are too low, or off-scale data, allelic/locus imbalance or other spurious artifacts when input template is too high (1-4). In addition, the laboratory is required to consume only what is needed to produce a result and retain the remaining evidence and/or DNA for independent testing by a different laboratory, should the court so order.
Standard 9.3 in the Quality Assurance Standards for Forensic DNA Testing Laboratories mandates that forensic DNA typing laboratories determine the amount of human genomic DNA recovered from samples (5). Any laboratory desiring accreditation/certification by accrediting agencies using these or other similar standards must quantitate human DNA recovered from biological evidence that is to be used for DNA typing.
Current non-specific methods available for DNA quantitation include the absorbance of ultraviolet light at 260 nm, quantitation of fluorescence in genomic DNA co-electrophoresed with known amounts of DNA in a “yield gel”, and quantitation of fluorescence produced by intercalating fluorescent dyes (6). Specific methods for quantitating human chromosomal DNA include quantitative hybridization of human DNA probes to slot blots or dot blots of dilutions of DNA from unknowns and quantitation standards (7), quantitative measurement of fluorescence produced by dyes intercalating in Alu sequences amplified from human genomic DNA (8,9), and fluorescence produced by accumulating DNA product measured after each extension step in a PCR cycling program (i.e. real-time PCR) (10-13). Real time PCR methods incorporate primers that target specific genomic sequences in human DNA whose accumulation during repeated rounds of amplification can be measured and is proportional to the amount of input DNA template. Included among human loci targeted for real time PCR are Alu sequences scattered throughout the genome (8) or sites on the X and Y chromosomes (11) whose amplification can provide data not only on DNA quantity, but also on the sex of the sample and the possible existence of male:female mixtures in extracts. Information regarding the gender of the person(s) contributing the DNA can be of vital importance, especially in the investigation of sexual assault cases. For example, if a forensic sample from a female contains a mixture of both male and female DNA, this could support an allegation of assault, whereas the absence of male DNA in a sample might argue against an assault having occurred.
Of the methods described above, only those that employ the use of human specific probes or primers will meet the intent of the quality assurance standards in that they will produce an estimate of human DNA quantity. However, to date, the methods of doing so involve the use of materials and instrumentation other than that which is required for PCR amplification of the DNA. This means that a laboratory that is engaged in DNA forensic analysis must either invest in the necessary equipment and training, or subcontract the work to other labs that are so equipped, both of which options are inefficient.
Another problem that plagues DNA analysts is that sometimes, in spite of a sufficiently high concentration of DNA in a sample, amplification of the DNA is inadequate due to the presence of PCR inhibitors. Such inhibitors may originate from clothing, dirt, etc. to which the sample has been exposed. While samples can be “cleaned up” to remove inhibitors, valuable sample and time may be wasted in detecting the presence of inhibitors, especially when samples are dealt with on a case by case basis.
The prior art has thus-far filed to provide a convenient method to, in a single reaction, analyze a DNA sample with respect to quantity, quality, presence of inhibitors, and gender of donor. In particular the prior art has failed to provide a method to obtain this information that requires only the use of equipment and personnel trained to carry out routine PCR, without additional instrumentation, supplies or training.